This invention relates generally to the field of medical diagnostics, and, more particularly, to a method for determining cell membrane dielectric breakdown and lymphocyte cell membrane fluidity in order to establish lymphocyte tumor-cell cytotoxicity as well as the presence of actual tumor-cells.
The lymphatic system of vessels in the vertebrate body begins in a network of exceedingly thin-walled capillaries in almost all the organs and tissues except the brain and bones. This network is drained by larger channels, mostly coursing along the veins and eventually joining to form a large vessel, the thoracic duct, which runs beside the spinal column to enter the left subclavian vein at the base of the neck. The lymph fluid originates in the tissue spaces by filtration from the blood capillaries, while in the lymphatic capillaries it is clear and watery. However, at intervals along the larger lymphatic vessels, the lymph passes through spongelike lymph nodes, where it receives great numbers of cells, the lymphocytes, and becomes turbid.
Lymphocytes are of different sizes; small, medium, and large. The range in size of these three kinds, however, varies with different classes and even species of animals. The large lymphocyte resembles very closely the stem cell of blood-forming tissue, the hemocytoblast. In fact, lymphoid tissue itself bears a considerable resemblance to hematopoietic tissue, and in lower vertebrates it is not always possible to distinguish one from the other.
In general, the lymphocyte is responsible for in vivo identification and removal of foreign, non-self material. Such material can be in the form of antigenic particulates, bacterial and virial bodies, and their associated soluble materials. The lymphocyte thus acts as a surveillance network primed to recognize and clear from the body's blood stream and tissues all potentially harmful irritants; it is the body's main line of defense and it is crucial to the maintenance of an organism's state of health.
There are several different types of lymphocytes required to accomplish the task of defending an organism against infection and disease. The following cell-types have been recognized based on their biological activity in vivo: T.sub.d cells are responsible for delayed-hypersensitivity reactions; T.sub.s cells are responsible for regulating cell-mediated immune reactions through suppression of other lymphocyte activities; T.sub.h cells are helper cells involved in antibody production; B lymphocytes are antibody producing cells; and T.sub.c lymphocytes are responsible for cell-mediated cytotoxicity reactions.
The T.sub.c lymphocyte functions as a killer cell towards invading tumor cells, bacteria, and virions, and its nomenclature, cytotoxic lymphocyte, derives from activity. These cells are crucial to thwarting foreign cell proliferation in vivo, and controlling and enhancing or potentiating their activity is a goal of chemical investigators interested in altering the balance of immune reactions in favor of the patient combating disease.
Accordingly, it is of major importance to determine whether an individual suspected of carrying a disease process, like a neoplastic or cancer cell, has functionally active T.sub.c lymphocytes. This information will reflect the patient's T.sub.c lymphocyte capacity to destroy the invading cell population.
A prior technique utilized to assay for cytotoxic activity of lymphocytes is set forth in, for example, the publication by John T. Harrington, Jr., entitled "Differing Time Courses of Spleen Leukocyte MIF Synthesis and Cytotoxicity During Rejection of a Murine Lymphoma Allograft," Cellular Immunology, Vol. 24, pgs 195-201, 1976 and described by Eric Martz, in the publication Contemporary Topics in Immunobiology, Vol. 7, pg 301, 1977. Such a technique is laborious, lengthy, expensive, and potentially hazardous. For example, the past procedure involves culturing lymphocytes in the presence of radioactively labeled tumor cells for approximately four hours under conditions of controlled temperature and humidity, and then measuring the cummulative release of radioactive radioisotope from the tumor cell as they are lysed or ruptured by the lymphocytes. This in vitro measurement of tumor cell death is believed to be correlated to in vivo cytotoxic activity of T.sub.c cells.
The actual events that occur during the process of cytotoxity are complicated and not well understood, but they presumably involve permeability changes in the tumor cell induced by cell contact with T.sub.c lymphocytes; this contact is essential and called the "lethal hit". The release of radioactive radioisotope from the tumor cell in this assay is believed to represent tumor cell death.
As stated above the prior technique is extremely lengthy in that tumor and lymphocytes must be prepared and cultured in an incubator for four hours. The assay involves the use of radioactive isotopes and requires strict safety and handling precautions. In addition, the incubation chamber, gas tanks, the radioactive isotope, and the well-geiger counter for quantitating radioactivity utilized with the past procedure are extremely expensive.
It is therefore essential in the field of medical diagnostics and more particularly in the field which encompasses the management of tumor-associated disease processes to obtain a reliable, fast, inexpensive in vitro alternative to the past technique in the quantification of lymphocytes tumor-cell cytotoxicity and lymphocyte membrane fluidity.